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Alexander van Oudenaarden. Pawlosky, Annalisa M. (Annalisa Marie) Harvard--MIT Program in Health Sciences and Technology. Harvard University--MIT Division of Health Sciences and Technology 2014-05-23T19:38:57Z 2014-05-23T19:38:57Z 2014 2014 http://hdl.handle.net/1721.1/87503 879668690 Thesis: Ph. D., Harvard-MIT Program in Health Sciences and Technology, 2014. Cataloged from PDF version of thesis. Includes bibliographical references. This work investigates the fundamental processes used by mammalian cells and organisms to make decisions during embryonic development. Current technologies that evaluate biological phenomenon often force a compromise between quantification of gene expression via bulk assays and qualitative imaging of cell and tissue heterogeneity. There are few options that allow for quantitative, high-resolution, single-cell analysis that is robust but not associated with a high degree of technical difficulty or obscured by amplification. Here, we address these issues using two model systems, the developing mammalian inner ear and single mouse embryonic stem cells (mESCs) during the process of X inactivation, to demonstrate our ability to perform single-cell, single-molecule assays that reveal both highly quantitative and spatial information. Accordingly, we adapted a high resolution, single-molecule RNA fluorescent in situ hybridization technique (smFISH) to study gene expression in the inner ear and perform allele-specific detection of the X chromosome in mESCs. We used previously-published smFISH procedures as our initial template for investigating biological signaling phenomena in these two systems. To study gene expression in the mouse inner ear, we developed a modified smFISH strategy to investigate mRNA transcript expression patterns in the cochlea during auditory hair cell development. The mammalian cochlea, a highly specialized and complex organ, beautifully demonstrates both the depth and breadth of the smFISH technique. To assay signaling behavior and topological changes of the X chromosome prior to X inactivation, we incorporated a novel allele-specific modification into the smFISH technique. We investigate the allele-specific expression patterns of eight genes that tile the X chromosome, which were chosen for their varied putative roles before, during and after X chromosome inactivation. Taken together, these two systems recapitulate the strength of the smFISH technique and its adaptations. The goals of this thesis were twofold: (1) expand the smFISH technique to work in specialized mammalian systems such as the cochlea and (2) demonstrate allele-specific DNA topological changes and expression patterns in mESCs. Elucidating high-resolution, single-molecule quantifiable imaging methods for application to complex tissues or allele-specific probing will have profound impacts on future investigations and promote a deeper comprehension of these systems. by Annalisa M. Pawlosky. Ph.D. 93 pages eng Massachusetts Institute of Technology M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission. http://dspace.mit.edu/handle/1721.1/7582 Harvard--MIT Program in Health Sciences and Technology. Single molecule techniques to probe decision-making processes in developmental biology Thesis application/pdf false Publication unknown unknown <Publication xmlns="https://www.openaire.eu/cerif-profile/1.1/" id="0e1f39b9-abfa-492e-af79-633af26f2e3d"> <Type xmlns="https://www.openaire.eu/cerif-profile/vocab/COAR_Publication_Types">http://purl.org/coar/resource_type/c_1843</Type> <Language>eng</Language> <Title>Single molecule techniques to probe decision-making processes in developmental biology</Title> <PublishedIn> <Publication> </Publication> </PublishedIn> <PublicationDate>2014</PublicationDate> <Authors> <Author> <DisplayName>Pawlosky, Annalisa M. (Annalisa Marie)</DisplayName> <Affiliation> <OrgUnit> </OrgUnit> </Affiliation> </Author> </Authors> <Editors> </Editors> <Publishers> <Publisher> <DisplayName>Massachusetts Institute of Technology</DisplayName> <OrgUnit /> </Publisher> </Publishers> <License>http://dspace.mit.edu/handle/1721.1/7582</License> <Keyword>Harvard--MIT Program in Health Sciences and Technology.</Keyword> <Abstract>This work investigates the fundamental processes used by mammalian cells and organisms to make decisions during embryonic development. Current technologies that evaluate biological phenomenon often force a compromise between quantification of gene expression via bulk assays and qualitative imaging of cell and tissue heterogeneity. There are few options that allow for quantitative, high-resolution, single-cell analysis that is robust but not associated with a high degree of technical difficulty or obscured by amplification. Here, we address these issues using two model systems, the developing mammalian inner ear and single mouse embryonic stem cells (mESCs) during the process of X inactivation, to demonstrate our ability to perform single-cell, single-molecule assays that reveal both highly quantitative and spatial information. Accordingly, we adapted a high resolution, single-molecule RNA fluorescent in situ hybridization technique (smFISH) to study gene expression in the inner ear and perform allele-specific detection of the X chromosome in mESCs. We used previously-published smFISH procedures as our initial template for investigating biological signaling phenomena in these two systems. To study gene expression in the mouse inner ear, we developed a modified smFISH strategy to investigate mRNA transcript expression patterns in the cochlea during auditory hair cell development. The mammalian cochlea, a highly specialized and complex organ, beautifully demonstrates both the depth and breadth of the smFISH technique. To assay signaling behavior and topological changes of the X chromosome prior to X inactivation, we incorporated a novel allele-specific modification into the smFISH technique. We investigate the allele-specific expression patterns of eight genes that tile the X chromosome, which were chosen for their varied putative roles before, during and after X chromosome inactivation. Taken together, these two systems recapitulate the strength of the smFISH technique and its adaptations. The goals of this thesis were twofold: (1) expand the smFISH technique to work in specialized mammalian systems such as the cochlea and (2) demonstrate allele-specific DNA topological changes and expression patterns in mESCs. Elucidating high-resolution, single-molecule quantifiable imaging methods for application to complex tissues or allele-specific probing will have profound impacts on future investigations and promote a deeper comprehension of these systems.</Abstract> <Access xmlns="http://purl.org/coar/access_right" > </Access> </Publication>